Abstract The equilibrium of aqueous D-glucaric acid was investigated via Nuclear Magnetic Resonance (NMR) spectroscopy. The NMR spectra of all four species (D-glucaric acid, D-glucaro-1,4-lactone, D-glucaro-6,3- lactone and D-glucaro-1,4;6,3-lactone) were assigned. A 1H NMR spectroscopy method was developed to investigate the kinetics of equilibration of the starting species (D-glucaro-1,4-lactone and D-glucaro-1,4;6,3-dilactone). The equilibration was investigated under neutral conditions as well as conditions with increasing acidity. Each experiment set contained 50-100 1HNMR spectroscopy experiments that were run on the same sample using a program that built in delays. Dimethyl sulfoxide was used as an internal standard, and its signal size was used as a scale to report the changes in relative concentration of the four species throughout the experiment sets. Under neutral conditions D-glucaro-1,4-lactone is relatively stable against equilibration, while D-glucaro-1,4;6,3-dilactone is not. Under acidic conditions both compounds equilibrate within approximately 30,000 seconds. After equilibration under acidic conditions D-glucaric acid is the dominant species, while the relative concentration of D-glucaro-1,4-lactone is slightly higher than that of D-glucaro-6,3-lactone. The relative equilibrium concentration of D-glucaro-1,4;6,3-dilactone is low. A mechanism for the equilibration of aqueous D-glucaric acid was proposed and equilibrium constants and estimates of rate constants were derived from the experimental data. These rate constants were used in MATLAB simulations that were compared to the experimental data. MATLAB simulations were used to alter the rate constants to improve the fits between experimental data and simulated data.

Several cell culture and animal studies demonstrated that citrus limonoids have protective effects against certain types of cancer. These chemopreventive properties of citrus limonoids are attributed to the induction of phase II enzyme, glutathione Stransferase (GST). In the current study, six citrus limonoids and two modified limonoids were utilized for the evaluation of NAD(P)H: quinone reductase (QR) activity and glutathione S-tranferase (GST) activity against 1-chloro-2,4-dinitrobenzene (CDNB) and 4-nitroquinoline 1-oxide (4NQO) in A/J female mice. In liver, limonoids that induced phase II enzyme activity were limonin-7- methoxime (32% CBNB), (270% 4NQO), (65% QR); and deacetylnomilin (180% QR). In stomach, limonin-7-methoxime (51% 4NQO); deacetyl nomilinic acid glucoside (55% 4NQO), nomilin (58% CDNB), (75% 4NQO); isoobacunoic acid (25% CDNB); deacetylnomilin (19% CDNB); limonoid mixture (45% 4NQO), (200% QR). Furthermore, in intestine, nomilin (280% 4NQO); deacetylnomilin (73% 4NQO), (22% QR); and the limonoid mixture (93% 4NQO) increase enzymatic activity. Finally in lung, deacetyl nomilinic acid glucoside (67% CDNB); limonin-7-methoxime (32% QR); and defuran limonin (45% QR) diplayed induction properties. Furthermore, D-glucaric acid (GA), a chemoprotective compound found in fruits and vegetables, was quantified using High Performance Liquid Chromatography (HPLC) grapefruit. Nine widely used grapefruit varieties were analyzed for the levels of D-glucaric acid. Seasonal levels of GA in each of the grapefruit varieties tested were found to be Thompson (58.36-126.8 mg/100ml), Henderson (29.6-49.7 mg/100ml), Rio Red (40.0-58.8mg/100ml), Star Ruby (25.5-46.7 mg/100ml), I-48 (26.6-58.3 mg/100ml), Ruby Red (49.3-63.0 mg/100ml), Ray’s Ruby (58.2-72.1 mg/100ml), Marsh (53.7-65.8 mg/100ml) and Duncan (50.17 mg/100ml). The HPLC method developed for the quantification of D-glucaric acid was found to be simple, fast, and reproducible. Additionally, the labor intensity and cost of sample preparation were greatly reduced.